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1.
PLoS One ; 9(8): e104878, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25137153

RESUMO

BACKGROUND: Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control. METHODOLOGY/PRINCIPAL FINDINGS: We conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production. CONCLUSIONS/SIGNIFICANCE: The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.


Assuntos
Aedes/enzimologia , Genoma de Inseto , Oviposição/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Vitelogeninas/genética , Aedes/efeitos dos fármacos , Aedes/genética , Sequência de Aminoácidos , Animais , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/enzimologia , Feminino , Regulação da Expressão Gênica , Himecromona/análogos & derivados , Himecromona/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ovário/efeitos dos fármacos , Ovário/enzimologia , Oviposição/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vanadatos/farmacologia , Vitelogeninas/antagonistas & inibidores , Vitelogeninas/biossíntese
2.
Development ; 136(17): 2987-96, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19666825

RESUMO

Tail regeneration in Xenopus tadpoles is a favorable model system to understand the molecular and cellular basis of tissue regeneration. Although turnover of the extracellular matrix (ECM) is a key event during tissue injury and repair, no functional studies to evaluate its role in appendage regeneration have been performed. Studying the role of Hyaluronan (HA), an ECM component, is particularly attractive because it can activate intracellular signaling cascades after tissue injury. Here we studied the function of HA and components of the HA pathway in Xenopus tadpole tail regeneration. We found that transcripts for components of this pathway, including Hyaluronan synthase2 (HAS2), Hyaluronidase2 and its receptors CD44 and RHAMM, were transiently upregulated in the regenerative bud after tail amputation. Concomitantly, an increase in HA levels was observed. Functional experiments using 4-methylumbelliferone, a specific HAS inhibitor that blocked the increase in HA levels after tail amputation, and transgenesis demonstrated that the HA pathway is required during the early phases of tail regeneration. Proper levels of HA are required to sustain proliferation of mesenchymal cells in the regenerative bud. Pharmacological and genetic inhibition of GSK3beta was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3beta is downstream of the HA pathway. We have demonstrated that HA is an early component of the regenerative pathway and is required for cell proliferation during the early phases of Xenopus tail regeneration. In addition, a crosstalk between HA and GSK3beta signaling during tail regeneration was demonstrated.


Assuntos
Ácido Hialurônico/metabolismo , Larva , Regeneração/fisiologia , Cauda/fisiologia , Xenopus laevis , Animais , Animais Geneticamente Modificados , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases , Ácido Hialurônico/genética , Himecromona/análogos & derivados , Himecromona/metabolismo , Larva/anatomia & histologia , Larva/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Transdução de Sinais/fisiologia , Cauda/anatomia & histologia , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/anatomia & histologia , Xenopus laevis/fisiologia
3.
Biochim Biophys Acta ; 1774(9): 1079-91, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17720633

RESUMO

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.


Assuntos
Glucana 1,4-beta-Glucosidase/metabolismo , Animais , Sítios de Ligação , Etildimetilaminopropil Carbodi-Imida/farmacologia , Glucana 1,4-beta-Glucosidase/antagonistas & inibidores , Glucanos , Glucosídeos/metabolismo , Gafanhotos/enzimologia , Concentração de Íons de Hidrogênio , Hidroximercuribenzoatos/farmacologia , Himecromona/análogos & derivados , Himecromona/metabolismo , Cinética , Masculino , Modelos Químicos , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos
5.
Mol Hum Reprod ; 6(8): 699-706, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10908279

RESUMO

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.


Assuntos
Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Animais , Células CHO , Cricetinae , Feminino , Humanos , Concentração de Íons de Hidrogênio , Himecromona/análogos & derivados , Himecromona/metabolismo , Himecromona/farmacologia , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/farmacologia
6.
Arch Microbiol ; 168(4): 321-7, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9297470

RESUMO

The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside. These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units. Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.


Assuntos
Celulase/metabolismo , Glicosídeo Hidrolases/metabolismo , Myxococcales/enzimologia , Myxococcales/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Carboximetilcelulose Sódica/metabolismo , Celobiose/análogos & derivados , Celobiose/metabolismo , Celulase/química , Celulase/isolamento & purificação , Celulose/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Quitosana , Cromatografia por Troca Iônica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Himecromona/análogos & derivados , Himecromona/metabolismo , Dados de Sequência Molecular , Myxococcales/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trissacarídeos/metabolismo
7.
Glycobiology ; 7(8): 1237-46, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9455925

RESUMO

Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to acceptors containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases. The trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids. Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli. We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme. The rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature. While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration. At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors. With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures. This finding is explained by an 8-fold increase in the Km for the acceptor from 13 degrees C to 33 degrees C. Differences in hydrolysis and transfer rates were also obtained by using 4-methylumbelliferyl-N-acetyl-neuraminic acid. However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed. In addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release. Based on these results we propose that transglycosylation in T. cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor. With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration.


Assuntos
Glicoproteínas/metabolismo , Neuraminidase/metabolismo , Temperatura , Trypanosoma cruzi/enzimologia , Animais , Sítios de Ligação , Catálise , Escherichia coli , Corantes Fluorescentes , Galactose/metabolismo , Glicosilação , Hidrólise , Himecromona/análogos & derivados , Himecromona/metabolismo , Lactose/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência
8.
Appl Environ Microbiol ; 58(9): 3095-100, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1444424

RESUMO

A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.


Assuntos
Toxinas Bacterianas/genética , Sondas de DNA , Enterotoxinas/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Himecromona/análogos & derivados , Tiogalactosídeos/metabolismo , Microbiologia da Água , Meios de Cultura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Água Doce , Genes Bacterianos , Himecromona/metabolismo , Oxigenases/metabolismo , Especificidade da Espécie , Virulência
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